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1 Architecture et Fonction des Macromolécules Biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 6098 and Universités d’Aix-Marseille I and II, 13402 Marseille Cedex 20, France
2 Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique (CNRS), UMR 6097, Sophia-Antipolis, 06560 Valbonne, France
3 Suntory Institute for Bioorganic Research, Mishima-Gun, Shimamoto-Cho, Wakayamadai 1–1–1, Osaka 618-8503, Japan
Reprint requests to: Hervé Darbon, AFMB, CNRS, UMR 6098 and Universités d’Aix-Marseille I and II, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France; e-mail: herve{at}afmb.cnrs-mrs.fr; fax: +33 (0)4-91-16-45-36; or Pierre Escoubas, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, UMR 6097, 660 Route des Lucioles, Sophia-Antipolis, 06560 Valbonne, France; e-mail: escoubas{at}ipmc.cnrs.fr; fax +33 (0)4-93-95-77-08.
(RECEIVED December 18, 2003; FINAL REVISION February 3, 2004; ACCEPTED February 3, 2004)
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Keywords: Phrixotoxin 1; NMR; spider toxin; structure determination; potassium channel gating modifier
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03584304.
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Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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). The progressive appearance of the final product was accompanied by the presence of a number of rapidly formed intermediates of undefined structure, which appeared to quantitatively decrease as the toxin folded properly. Over the course of 72 h, most of the linear peptide folded into its expected conformation, and the refolding reaction was terminated as conversion of the remaining intermediate forms appeared to become extremely slow. Final purification yielded more than 3 mg of synthetic PaTx1. Coelution experiments, mass spectrometry, and activity assays on heterologously expressed Kv4 channels demonstrated complete identity with the native toxin isolated from spider venom (data not shown). Refolding of ICK peptides has often been problematic in our hands (P. Escoubas, unpubl.) and as reported by others (DeLa Cruz et al. 2003) yielding very low amounts of correctly folded toxins. Various methods have been used to modify or supplement the refolding buffer composition to increase yields (Kubo et al. 1996; DeLa Cruz et al. 2003). We have found in the case of PaTx1 that refolding at 4°C permitted the conversion of folding intermediates into the final product with good efficiency, and in this case at least, refolding proceeded well with a good yield. When compared to other ICK peptides of different structure, this might be linked to the shorter length of PaTx1 or the reduced flexibility of its intercysteine loops.
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). The repartition of the H
i/HNi+1, H
i/HNi+1, and HNi/HNi+1 correlations indicates that the toxin is organized in loops, beside three short extended regions characterized by strong Hi/HNi+1 correlations together with large coupling constants (Fig. 2A).
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. In addition, 18 hydrogen bond restraints derived from proton exchange and 13 dihedral angle restraints derived from the measurement of coupling constants were included, as well as 9 distance restraints derived from the three disulfide bridges that were assigned by homology with related spider toxins (Escoubas et al. 2000b). Altogether, the final experimental set corresponded to 12.3 constraints per residue on average.
The distance geometry calculation using the whole set of restraints and energy minimization led to a single family of 25 structures (Fig. 3A,B) and the structural statistics are given in Table 2. The root mean square deviation (RMSD) calculated on the whole structure is 1.05 ± 0.29 Å for the backbone and 2.10 ± 0.34 Å for all heavy atoms. If residues 2–28 only are taken into account, these values become 0.71 ± 0.18 Å and 1.68 ± 0.28 Å, respectively, which indicates a poor resolution of N- and C-terminal residues. This is confirmed by the individual RMSD values (Fig. 2B). This low resolution, in particular for the C-terminal part of the toxin is experimentally due to the absence of structural restraints and can be explained by the high mobility of these residues in solution (Fig. 3A).
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The analysis of the Ramachandran plot for the ensemble of the 25 calculated models (in PROCHECK software nomenclature) reveals that 58.9% of the residues are in the most favored regions, 31.4% in the additional allowed regions, 5.2% in the generously allowed regions, and 4.5% in the disallowed regions (data not shown). The only residue that is found constantly in a disallowed region is Leu 23, which is in a 
-turn conformation: The average angle values for the 25 solutions are 80.6 ± 4.0 for angle 
and –54.4 ± 8.3 for angle 
and a hydrogen bond connects the carboxyl oxygen of Arg 22 and the amide proton of Trp 24. The conformation of this turn present in all ICK toxins depends upon its length, in some toxins described as a -turn, and in others as a undefined loop (see Fig. 4).
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shows a stereopair representation of the best-fit superimposition of the backbone traces of the 25 best structures and Figure 3B shows a MOLSCRIPT representation of the average structure.
The existence of H19-H27, H19-HN28, and H27-HN20 nOe together with the upfield shift of the HN protons of residues 20 and 26 compared to random coil values and associated with large H-HN coupling constants and slowly exchanging HN protons for residues 19, 20, and 26 indicated a short two-stranded antiparallel -sheet as the only secondary structure. It includes residues 19–20 for the first strand and residues 26–27 for the second strand. The two strands are well detected by the PROCHECK-NMR software. A third extended structure is observed between residues Trp 7 and Cys 9. Hydrogen bonds are detected between the carboxyl oxygen of Trp 7 and the amide proton of Cys 25, and between the amide proton of Cys 9 and the carboxyl oxygen of Leu 23 (Fig. 3B). This third extended strand has been similarly described in several other ICK toxins (Mosbah et al. 2000).
All the amide protons of PaTx1 resonate at conventional frequencies with the exception of Lys 4, which shows an unusual chemical shift for its amide proton (i.e., 5.01 ppm at 290 K, 5.19 ppm at 300 K, and 5.25 ppm at 310 K). Determination of the structure of PaTx1 gives an explanation of this unusual chemical shift: The amide proton is placed in the neighborhood of the aromatic ring of Trp 7 as shown in Figure 3C. The ring current of Trp7 creates an electromagnetic shield that dramatically affects most of the chemical shifts of the Lys 4 protons (Table 1). This is also observed for the chemical shifts of Leu 23, similarly affected by the ring current effect of Trp 24.
Comparison with related toxins
The fold of PaTx1 places it in the inhibitor cystine knot (ICK) peptide family (Craik et al. 2001). The ICK fold family includes numerous toxic and inhibitory peptides from animal venoms. Many ion channels effectors from marine snails, spider, and scorpion venoms share this fold, although this common molecular scaffold supports a wide diversity of pharmacological profiles. Toxins folded according to the ICK motif can demonstrate varying specificity against different subtypes of voltage-dependent calcium (Mosbah et al. 2000; Bernard et al. 2001), sodium (Omecinsky et al. 1996), or potassium channels. In spider toxins, the ICK fold has also been associated with inhibition of acid-sensing ion channels (ASICs; Escoubas et al. 2000a, 2003). Among potassium channel toxins, PaTx1 and its isoform PaTx2 are active on Kv4.2 and Kv4.3 potassium channels, 
-conotoxin PVIIA from the cone snail Conus purpurascens acts on shaker channels (Scanlon et al. 1997; Savarin et al. 1998; Shon et al. 1998), the hanatoxins HaTx1 and HaTx2 act on Kv2.1 channels (Swartz and MacKinnon 1995; Takahashi et al. 2000; Lou et al. 2002), and the heteropodatoxin HpTx2 is active on Kv4.2 (Sanguinetti et al. 1997; Bernard et al. 2000). We have also recently described novel toxins from the venom of the theraphosid spiders Stromatopelma calceata (ScTx1) and Heteroscodra maculata (HmTx1 and HmTx2), which inhibit different subtypes of both Kv2 and Kv4 channels (Escoubas et al. 2002). Common features of these peptides are their activity against voltage-dependent channels, related amino acid sequences with highly conserved cysteine arrangement (Fig. 4B), and a similar ICK scaffold with three disulfides bridges and a two- or three-stranded antiparallel -sheet (Fig. 4A).
As the ICK scaffold can bear a variety of pharmacological functions, careful analysis of the molecular surface of the toxin and delineation of a putative functional surface is of utmost importance to understand how subtle variations of the primary sequence influence the spatial structure of the toxin and hence define a specific pharmacology. With that aim, our group has developed a prediction method of the molecular dipole moment which reveals the electrostatic anisotropy of peptides. The molecular surface through which the dipole emerges is hypothesized to be involved in the interaction with the receptor site of the toxin (Blanc et al. 1997; Fremont et al. 1997; Ferrat et al. 2001). This prediction method appears to work well for toxins acting by pore occlusion, for which structure–activity data based on site-directed mutagenesis is available. The predicted interaction surface is confirmed by the reduction in biological activity in series of residue analogs (Sabatier et al. 1994; Inisan et al. 1995; Fremont et al. 1997).
For PaTx1, the overall charge distribution reveals a marked electrostatic anisotropy that is represented by the dipole moment shown in Figure 5A. The electrostatic calculation shows that this dipole emerges through the -sheet (near the Lys 26) defining a potential interaction surface composed of three basic residues (Lys 26, Lys 27 in the antiparallel -sheet, and Arg 22 in the turn), two aromatic residues (Trp 24, Trp 5) and two hydrophobic amino acids (Leu 23 and Ile 28).
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. Interestingly, the dipole moment of PaTx1 emerges through Lys 26 similarly to the dipole of HpTx2, which emerges through Lys 27. These two toxins, both isolated from spider venom, although from different families, act on the same channel (Kv4.2) and present a very similar putative surface. Close to the central lysine, another basic residue is present (Arg 22 or Arg 23 for PaTx1 and HpTx2, respectively), as well as an aromatic cluster (Trp 5 and Trp 24 for PaTx1, Phe 7, Trp 25, and Trp 30 for HpTx2) and an aliphatic side chain (Leu 23 or Leu 24 for PaTx1 and HpTx2, respectively). At first glance, these surfaces appear similar to those of other Kv channel inhibitors that occlude the extracellular entryway of the pore, such as conotoxin -PVIIA (Shon et al. 1998). In those toxins, a critical lysine residue is involved in pore blocking and is always adjacent to an aromatic residue such as tryptophan or phenylalanine, forming a "critical dyad" as first defined by Dauplais et al. (1997).
However, PaTx1 has been described as a gating modifier (Diochot et al. 1999), and affects channel function by modulating voltage-dependent gating and not by pore occlusion. Similarly, hanatoxin 1, another KV channel gating modifier, acts via binding to the voltage-sensing region of the Kv2.1 channel, on the extracellular loop between segments S3 and S4 (Swartz and MacKinnon 1995Swartz and MacKinnon 1997a, b,Li-Smerin and Swartz 2001), thus affecting transmembrane movement of the voltage-sensing domain. The three-dimensional structure of HaTx1 has been solved by NMR (Takahashi et al. 2000) and a putative interaction surface was proposed, comprising a large hydrophobic patch (residues Tyr 4, Leu 5, Phe 6, Tyr 27, Ala 29, and Trp 30) surrounded by basic residues (Lys 22, Arg 24, and Lys 26; Fig. 5B).
In comparison with the three-dimensional structures of other gating-modifier toxins (Cse-V and ATX III) Takahashi et al. (2000) proposed that the putative interactive surface of HaTx1 could be homologous to the surface of Cse-V and ATX III. Indeed these peptides present a large hydrophobic patch (composed of five tyrosines for Cse-V and one tyrosine, two prolines, and two tryptophans for ATX III) surrounded by basic residues (Lys 13 and Lys 55 for Cse-V and Arg 1 and Lys 26 for ATX III). The dipole moment calculation applied to HaTx1 shows that the dipole emerges near Lys 26, through a surface composed of basic residues (Arg 24 and Lys 10), as well as aromatic amino acids (Phe 23 and Tyr 27) and an acidic residue (Asp 25). This point of emergence of the dipole moment is perpendicular to the hydrophobic patch described by Takahashi et al. The same calculation applied to Cse-V and ATX III shows that the dipole moment also emerges through the basic residues Arg 1 and Lys 26 for ATX III; Lys 13 and Lys 55 for Cse-V, perpendicular to the hydrophobic patch (data not shown). In Cse-V, these residues are considered essential for the toxicity (Jablonsky et al. 1995).
All these toxins acting on voltage-gated channels thus appear to possess a similar surface pattern, comprising a hydrophobic patch surrounded by basic residues from which the calculated dipole moment emerges. The structure of PaTx1 identifies the same pattern: The dipole moment emerges through Lys 26, perpendicular to Lys 26 and Lys 27, and is associated with a hydrophobic patch composed of Met 6, Trp 5, Trp 7, and Trp 24. For the couple HaTx1/ Kv2.1, the accepting surface on the channel has been determined by site-directed mutagenesis to involve the residues Ile 273, Phe 274, Glu 277, Arg 290, and Arg 296 (Swartz and MacKinnon 1997b). Both hydrophobic and electrostatic interaction would therefore take place at the toxin/channel interface, in accordance with our hypothesis.
To complete our study and verify whether the same model could apply to other toxins modulating Kv channels, we modeled both PaTx2, an isoform of PaTx1 also isolated from Phrixotrichus auratus venom (Diochot et al. 1999), and ScTx1 isolated from the venom of Stromatopelma calceata (Escoubas et al. 2002). PaTx2 possesses a high sequence similarity with PaTx1, differing only by two acidic residues at positions 11 and 12 and two C-terminal residues. PaTx2 is mainly active on Kv4.2 and Kv4.3, whereas ScTx1 is active on Kv2.1 and Kv2.2 and is the most potent inhibitor known to date for the Kv4.2 channel (IC50 = 1.2 nM). These two toxins are known to belong to the gating-modifier family, and their characteristics of channel inhibition are very similar to those of HaTx1, which blocks Kv2.1 and Kv4.2 channels.
The calculation of the dipole moment for the PaTx2 model shows that the dipole emerges from the -sheet but that the point of emergence is somewhat different when compared to the dipole of PaTx1. It emerges through Arg 27 (through Lys 26 in PaTx1), defining a surface composed of a basic residue (Arg 27), aromatic residues (Trp 5, Ile 28, and Ile 29) and an acidic residue (Glu 17). Similar to PaTx1, the dipole is perpendicular to the hydrophobic patch composed of Met 6, Trp 5, Trp 7, and Trp 24 (Fig. 5A). The difference between the residues from which the dipole moment emerges (Arg 27 for Patx2 and Lys 26 for PaTx1) and the additional presence of an acidic residue could provide an explanation for the reduced affinity of PaTx2 for Kv4.2 (IC50 = 5 nM for PaTx1 and IC50 = 34 nM for PaTx2) and Kv4.3 (IC50 = 28 nM for PaTx1 and IC50 = 71 nM for PaTx2).
For ScTx1, the dipole emerges through Lys 26, from a surface comprising a basic residue (Lys 26) as well as aromatic ones (Phe 6, Ala 8, and Tyr 27; Fig. 5A). In HaTx1, the toxin surface perpendicular to the dipole moment orientation is composed of residues Met 5, Phe 6, Tyr 27, Ala 29, and Trp 30. In the model of ScTx1, no acidic residue is present in the surface from which the dipole moment emerges, in contrast to HaTx1. This feature is perhaps to be correlated with the different affinity of ScTx1 and HaTx1 for the Kv2.1 channel (IC50 = 12.7 nM for ScTx1 and IC50 = 42 nM for HaTx1) and its much higher affinity for Kv4.2.
The sequences of ScTx1, HaTx1, HmTx1, PaTx1, PaTx2, and HpTx2 show conservation of an aromatic residue before the last cysteine (Fig. 4B): tyrosine for ScTx1, HaTx1, and HmTx1, tryptophan for PaTx1, PaTx2, and HpTx2. In HmTx2, a structural analog of HmTx1, this aromatic amino acid is replaced by a hydrophobic residue (Ile instead of Tyr) and unlike HmTx1, HmTx2 has weak activity against Kv2.1 and is devoid of activity on Kv2.2, Kv4.1, Kv4.2, and Kv4.3 (Escoubas et al. 2002). The superposition of the C trace of the six toxins shows that these aromatic residues are placed in the same locus, and that the basic residue from which the dipole emerges is located nearby. The conservation of a hydrophobic residue in a position close to a basic residue may therefore be of importance for the interaction with the target channel, forming a functional dyad. The calculated model of the toxin HmTx1 also possesses the same overall pattern of a hydrophobic patch and basic residues but the calculation of the dipole moment of this protein shows that the dipole emerges through the hydrophobic patch and not through the basic residue (data not show). This result has to be interpreted in the light of the different activity of this toxin when compared with HaTx1, in spite of their sequence similarity.
We therefore propose that the conserved orientation of the dipole moment that emerges through or in the immediate vicinity of a basic residue, combined with the presence of a hydrophobic patch could delineate the interaction surface of the toxin with its receptor. The dipole moment of the toxins would help orient the toxin in the electrostatic field of the channel, and binding could result from the combined interaction of the hydrophobic patch and the basic residues from which the dipole emerges, with complementary residues on the channel. Although dipole moments calculated on all so far known gating modifiers show a similar orientation, subtle differences in dipole orientation and toxin surface could affect both the dipole and the charge distribution on the surface, inducing the observed differences in the activity of PaTx1, HaTx1, ScTx1, and HmTx1. To check whether the electrostatic dipole of a gating modifier toxin may act in the same way as for pore blockers, we analyzed the charge distribution of the receptor domain, that is, the S3-S4 segment of Kv4.2. We modeled this segment on the basis of the KvAP X-ray structure (Jiang et al. 2003). The S3 helix is mainly acidic whereas the S4 helix is rich in basic residues. As a consequence, the resulting local dipole is oriented from S3 toward S4 and therefore could play a role in the orientation of the gating modifier toxins with regard to their receptor site.
Based on the above, our results using the dipole moment prediction model appear to be consistent with the contact surface of the voltage-gating modifier toxins, proposed by others (Takahashi et al. 2000). While this work was submitted for publication, an interaction model of Kv gating modifier toxins with the S3–S4 segment of Kv channels was proposed, outlining the same interaction surface (Shiau et al. 2003). However, the structure of mammalian Kv channels is still the objet of controversy, as descriptions of the structures of the KCSA and KvAP channels yielded contradictory results (Jiang et al. 2003). In the recently described KvAP structure, the S3–S4 segment would be buried in the lipid bilayer, a feature difficult to reconcile with the extracellular action of peptide toxins.
We therefore believe that until further work has been conducted to delineate the exact toxin channel contact surface for both hanatoxin type and phrixotoxin type Kv channel gating modifiers, docking modeling remains speculative. Site-directed mutagenesis of PaTx1 will shed additional light on the toxin-receptor binding mechanism on Kv4.2, and confirm the hypothesis of the interacting surfaces based on our structural investigations.
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MALDI-TOF mass spectrometry
All mass spectra were acquired on a Voyager DE-Pro instrument (Applied Biosystems) in either linear or reflector positive mode using -cyano-4-hydroxycinnamic acid (-CHCA) as the matrix (5 mg/mL). Spectra were calibrated externally (HPLC fractions) or internally (pure peptides) using standard peptide mixtures. Spectra were processed in Data Explorer, and all mass calculations were done using the GPMAW software (http://www.protein.sdu.dk/gpmaw/index.html).
Sample preparation for NMR
We solubilized 2.6 mg of pure synthetic PaTx1 in 500 µL of a H2O/D2O mixture (9:1 v/v) to give a concentration of 1.5 mM at pH 3.0. The amide proton exchange rates were obtained on a sample solubilized in 500 µL of 100% D2O.
NMR experiments
The 1H NMR spectra were all recorded on a BRUKER DRX500 spectrometer equipped with an HCN probe and self-shielded triple axis gradients were used. The experiments were performed at three different temperatures, 290, 300, and 310 K, to solve assignment ambiguities. Two-dimensional spectra were acquired using the States-TPPI method (Marion et al. 1989) to achieve F1 quadrature detection (Marion and Wüthrich 1983). Water suppression was obtained either using presaturation during the 1.5-sec relaxation delay and during the mixing time for NOESY spectra or with a watergate 3–9–19 pulse train (Piotto et al. 1992) using a gradient at the magic angle obtained by applying simultaneous x, y, and z gradients prior to detection. TOCSY were performed with a spin-lock time of 80 msec and a spin-locking field strength of 8 kHz. The mixing time of the NOESY experiments was set to 80 msec. The individual amide proton exchange rates were determined by recording two series of eight NOESY spectra (each experiment was 5 h long) at 290 and 300 K on the D2O sample. Amide protons still giving rise to nuclear Overhauser effect (nOe) correlations after 40 h of exchange were considered as slowly exchanging and therefore engaged in a hydrogen bond.
Analysis of spectra
The identification of amino acid spin systems and the sequential assignment were done using the standard strategy described by Wüthrich (1986) and regularly used by our group, with the XEASY graphical software (Eccles et al. 1991). The comparative analysis of COSY and TOCSY spectra recorded in water gave the spin system signatures of the protein. The spin systems were then sequentially connected using the NOESY spectra.
Experimental restraints
The integration of nOe data was done by measuring the peak volumes using a routine of XEASY. These volumes were then translated into upper limit distances by the CALIBA routine of the DIANA software (Günter et al. 1991). The lower limit distance was systematically set at 0.18 nm.
The 
torsion angle constraints resulted from the 3JHN-H coupling constant measurements that were measured on a COSY spectrum with 8192 data points in the acquisition dimension. These angles were restrained to –120 ± 40° for a 3JHN-H 
8 Hz and to –65 ± 25° for a 3JHN-H 
6 Hz. No angle constraint was assigned to a 3JHN-H = 7 Hz, a value considered as ambiguous.
Determination of the amide proton exchange rates led us to identify protons involved in hydrogen bonding. The oxygen partners were then identified by visual inspection of the preliminary calculated structures.
Structure calculation
The structures were calculated by the variable target function program DIANA 2.8 (Güntert et al. 1991). A preliminary set of 1000 structures was initiated including only intraresidual and sequential upper limit distances. From these, the 500 best (as judged by the absence of significant nOe violations) were kept for a second round, for which the restraints belonging to the 3 strands were injected together with the distance restraints defining the disulfide bridges (i.e., dS-S 0.21 nm, dC-S and dS-C 0.31 nm) and the hydrogen bonds included in the strands. The resulting 250 best solutions were selected for a third calculation run, including the whole set of upper limit restraints. The 50 best structures were finally refined by including the dihedral constraints together with the additional distance restraints coming from hydrogen bonds outside the sheet.
Then, to remove bad van der Waals contacts, these 50 structures were refined by restrained molecular dynamics annealing at 1000 K (parameters file: protein-allhdg), followed by slow cooling and energy minimization (10 cycles of 1000 steps each) in the CNS software (Brunger et al. 1998).
The visual analysis of the final selection of structures was carried out with the TURBO graphic software (Roussel and Cambillau 1989) and the geometric quality of the resulting structures was assessed with the PROCHECK 3.3 and PROCHECK-NMR software (Laskowski et al. 1996).
Electrostatic calculation
The electrostatic potential and dipole moment of the toxin were calculated using the GRASP software (Nicholls et al. 1991) running on a Silicon Graphics Workstation. This calculation includes all ionizable groups in the peptide, based on the amber force field of the residues. The potential maps were calculated with a simplified Poisson–Boltzmann solver (Nicholls and Honig 1991; Nicholls et al. 1991).
Molecular modeling
Molecular modeling of the structures of PaTx2, ScTx1, and HmTx1 was achieved using the published NMR structures coordinates of PaTx1 (for PaTx2) and HaTx1 (for ScTx1 and HmTx1). The structures were calculated using the MODELLER6 software: For each of the measured NMR conformations of a given template, we calculated five models and, based on the lowest MODELLER restraints energy, we kept only the best one. Our final structure was therefore based on 20 models for ScTx1 and HmTx1 and 25 models for PaTx2.
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